HHcy activates phospholipid hydrolysis of platelet membrane and ATX secretion. (A-E) HPLC-MS/MS analysis of lipid metabolites in platelets from C57BL/6J mice that were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline) for 2 hours. (A) Principal component analysis (PCA) scatterplot of lipid metabolites in platelets. (B) VIP scatterplot identified by PCA showing the top 15 lipid metabolites in the different groups. (C) Heat map illustrating the phospholipid metabolic profiles in platelets. (D) HPLC-MS/MS analysis of different levels of LPC species in platelets. (E) cPLA2 activity of platelets from acute HHcy and control mice in vivo. (F-H) C57BL/6J mice were intraperitoneally injected with 100 mg/kg Hcy or vehicle (saline) for 2 hours. Plasma ATX (F) and LPA (G) levels were measured via enzyme-linked immunosorbent assay (ELISA; Cloud-Clone Corp). (H) Western blot analysis of ATX expression in platelets. β-Actin was used as an internal control. (I-K) Washed platelets from C57BL/6J were incubated with 100 μM Hcy for 10 minutes, then stimulated with 1.5 μg/mL collagen. The levels of ATX (I) and LPA (J) in platelet-activated supernatant were measured by ELISA. ATX protein expression (K) in platelets was analyzed by western blot, with β-actin used as the internal control. All data are expressed as the mean ± SEM (n = 3-4). *P < .05 compared with ctrl; #P < .05 compared with collagen. (L-M) Correlations between human plasma Hcy concentrations and the levels of ATX (L) and LPA (M) were measured by ELISA (n = 88).